U.S. patent application number 12/999045 was filed with the patent office on 2011-04-28 for anti-prion protein antibody fragment.
This patent application is currently assigned to Carsten Korth. Invention is credited to Carsten Korth, Sirik Rutger Leliveld, Andreas Muller-Schiffmann, Benjamin Petsch, Lothar Stitz.
Application Number | 20110098448 12/999045 |
Document ID | / |
Family ID | 39951614 |
Filed Date | 2011-04-28 |
United States Patent
Application |
20110098448 |
Kind Code |
A1 |
Korth; Carsten ; et
al. |
April 28, 2011 |
ANTI-PRION PROTEIN ANTIBODY FRAGMENT
Abstract
The invention relates to an antibody, antibody fragment or
derivative thereof which specifically recognizes a prion protein
and which comprises a complementarity determining region (CDR), a
retro-inverso D-peptide of said CDR and/or an anti-idiotypic
antibody, antibody fragment or derivative thereof which recognizes
said CDR. The invention further concerns a nucleic acid molecule
encoding said antibody, antibody fragment or derivative thereof as
well as a method for generating an antibody, antibody fragment or
derivative thereof that specifically recognizes a prion protein,
wherein an antibody that recognizes a specific domain of the prion
protein is generated, an antigen-specific amino acid sequence is
isolated from said antibody, and an anti-idiotypic antibody,
antibody fragment or derivative thereof, which recognizes said
antigen-specific amino acid sequence, is generated.
Inventors: |
Korth; Carsten; (Dusseldorf,
DE) ; Stitz; Lothar; (Kiebingen, DE) ; Petsch;
Benjamin; (Tubingen, DE) ; Muller-Schiffmann;
Andreas; (Odenthal, DE) ; Leliveld; Sirik Rutger;
(Dusseldorf, DE) |
Assignee: |
Korth; Carsten
Dusseldorf
DE
Stitz; Lothar
Kiebingen
DE
Petsch; Benjamin
Tubingen
DE
Muller-Schiffmann; Andreas
Odenthal
DE
|
Family ID: |
39951614 |
Appl. No.: |
12/999045 |
Filed: |
June 16, 2009 |
PCT Filed: |
June 16, 2009 |
PCT NO: |
PCT/EP09/04358 |
371 Date: |
January 18, 2011 |
Current U.S.
Class: |
530/387.2 ;
530/389.4; 536/23.53 |
Current CPC
Class: |
A61P 25/28 20180101;
C07K 14/47 20130101; A61K 2039/505 20130101; C07K 2317/565
20130101; C07K 7/08 20130101; C07K 2317/34 20130101; C07K 16/2872
20130101; C07K 2317/622 20130101 |
Class at
Publication: |
530/387.2 ;
530/389.4; 536/23.53 |
International
Class: |
C07K 16/42 20060101
C07K016/42; C07K 16/08 20060101 C07K016/08; C07H 21/00 20060101
C07H021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 17, 2008 |
EP |
08010971.3 |
Claims
1. An antibody or antibody fragment specifically recognizing a
prion protein, comprising at least one amino acid sequence selected
from a group consisting of a complementarity determining region
(CDR) according to SEQ ID NO:1, a complementarity determining
region (CDR) according to SEQ ID NO:11, a complementarity
determining region (CDR) according to SEQ ID NO: 12, a
complementarity determining region (CDR) according to SEQ ID NO:
13, a retro-inverso D-peptide of said CDR according to SEQ ID NO:2,
and an anti-idiotypic antibody or antibody fragment, which
recognizes said CDR, comprising SEQ ID NO:3.
2. The antibody or antibody fragment according to claim 1, wherein
the complementarity determining region (CDR) is contained in at
least one heavy chain variable region according to SEQ ID NO:4.
3. The antibody or antibody fragment according to claim 2, further
comprising at least one light chain variable region according to
SEQ ID NO:5.
4. The antibody or antibody fragment according to claim 3, wherein
the at least one heavy chain variable region and the at least one
light chain variable region are linked by a linker peptide.
5. The antibody or antibody fragment according to claim 1, further
comprising at least one signal and/or tag sequence.
6. The antibody or antibody fragment according to claim 1,
comprising the amino acid sequence according to SEQ ID NO:6.
7. An antibody or antibody fragment, the amino acid sequence of
which is at least 85% identical to the amino acid sequence of the
antibody or antibody fragment according to claim 1, or with which
equivalent charged, hydrophobic, aromatic or alcoholic amino acids
are replaced within each other.
8. A nucleic acid molecule selected from a group consisting of a.
nucleic acid molecules encoding an antibody or antibody fragment
comprising the amino acid sequence according to SEQ ID NO:1; b.
nucleic acid molecules encoding an antibody or antibody fragment
comprising the amino acid sequence according to SEQ ID NO:2; c.
nucleic acid molecules encoding an antibody or antibody fragment
comprising the amino acid sequence according to SEQ ID NO:3; d.
nucleic acid molecules encoding an antibody or antibody fragment
comprising the amino acid sequence according to SEQ ID NO:4; e.
nucleic acid molecules encoding an antibody or antibody fragment
comprising the amino acid sequence according to SEQ ID NO:5; f.
nucleic acid molecules encoding an antibody or antibody fragment
comprising the amino acid sequence according to SEQ ID NO:6; g.
nucleic acid molecules encoding an antibody or antibody fragment
comprising the amino acid sequence according to SEQ ID NO:11; h.
nucleic acid molecules encoding an antibody or antibody fragment
comprising the amino acid sequence according to SEQ ID NO: 12; i.
nucleic acid molecules encoding an antibody or antibody fragment
comprising the amino acid sequence according to SEQ ID NO: 13; j.
nucleic acid molecules encoding the antibody or antibody fragment
according to claim 1; k. nucleic acid molecules comprising the
nucleotide sequence according to SEQ ID NO:7; l. nucleic acid
molecules comprising the nucleotide sequence according to SEQ ID
NO:8; m. nucleic acid molecules comprising the nucleotide sequence
according to SEQ ID NO:9; n. nucleic acid molecules comprising the
nucleotide sequence according to SEQ ID NO:10; o. nucleic acid
molecules, the polynucleotide sequence of which is at least 85%
identical to the nucleotide sequence of a nucleic acid molecule of
any of a) to n), and which encode an antibody or antibody fragment
that specifically recognizes a prion protein; p. nucleic acid
molecules, the complementary strand of which hybridizes to a
nucleic acid molecule of any of a) to n), and which encode an
antibody or antibody fragment that specifically recognizes a prion
protein; q. nucleic acid molecules, the nucleotide sequence of
which differs from the nucleotide sequence of a nucleic acid
molecule of any of a) to p) due to the degeneracy of the genetic
code; and r. nucleic acid molecules, the nucleotide sequence of
which is complementary to the nucleotide sequence of a nucleic acid
molecule of any of a) to q).
9. A method for generating an antibody or antibody fragment that
specifically recognizes a prion protein, the method comprising: a)
generating an antibody that recognizes a specific domain of the
prion protein; b) isolating an antigen-specific amino acid sequence
from said antibody; and c) generating an anti-idiotypic antibody or
antibody fragment, which recognizes said antigen-specific amino
acid sequence.
10. The method according to claim 9, wherein the specific domain is
an interaction domain of the prion protein.
11. The method according to claim 9, wherein the antigen-specific
amino acid sequence is a complementarity determining region (CDR)
or a retro-inverso sequence of said CDR.
12. A kit comprising the antibody or antibody fragment according to
claim 1.
13. A pharmaceutical preparation comprising the antibody or
antibody fragment according to claim 1.
14-15. (canceled)
16. The antibody or antibody fragment according to claim 4, wherein
the linker peptide is (Gly.sub.4Ser).sub.3.
17. The antibody or antibody fragment according to claim 7, wherein
the amino acid sequence of which is at least 90% identical to the
amino acid sequence of the antibody or antibody fragment according
to claim 1, or with which equivalent charged, hydrophobic, aromatic
or alcoholic amino acids are replaced within each other.
18. The antibody or antibody fragment according to claim 7, wherein
the amino acid sequence of which is at least 95% identical to the
amino acid sequence of the antibody or antibody fragment according
to claim 1, or with which equivalent charged, hydrophobic, aromatic
or alcoholic amino acids are replaced within each other.
Description
FIELD OF THE INVENTION
[0001] The invention relates to an antibody or antibody fragment
which recognizes a prion protein. The invention further relates to
a nucleic acid molecule encoding the antibody or antibody fragment
and a method for generating the antibody or antibody fragment.
BACKGROUND OF THE INVENTION
[0002] Prion diseases are unique, transmissible, neurodegenerative
diseases since the infectious agent consists solely of an
alternative conformational isoform of the host-encoded prion
protein, PrP.sup.Sc, that replicates without a nucleic acid
(Prusiner, 1982; Prusiner, 1998; Safar et al., 2005). Replication
is thought to occur by induction of the infectious conformation in
the normal prion protein PrP.sup.C (Prusiner, 1982). The different
stable conformations, or "conformers", of PrP have pioneered the
concept of protein conformational diseases within the
neurodegenerative diseases stating that a misfolded or misprocessed
protein is causative in the pathogenesis of the disease (Prusiner,
2001; Taylor et al., 2002). While due to the insolubility of many
of the misfolded proteins, structural analysis has been difficult,
generation of ligands specific for the misfolded proteins has been
key to analyze these protein conformations in their cellular
environment (Leliveld and Korth, 2007). The notion that soluble
alternatively folded conformers or oligomers of proteins rather
than insoluble protein deposits are instrumental in the disease
processes has focussed efforts to develop conformer- or
oligomer-specfic ligands. Conformation-specific monoclonal
antibodies (mABs) have been raised to PrP.sup.Sc (Korth et al.,
1997; Paramithiotis et al., 2003) or to A.beta. oligomers which are
major pathogenic conformers in Alzheimer disease (Kayed et al.,
2003), enabling detection of single conformers of proteins within a
population of proteins. These reagents have become key reagents in
detecting presence of these disease-associated conformers in
tissues or body fluids as a method of identifying asymptomatic or
early stage individuals at risk to developing prion disease (Kuhn
et al., 2005; Nazor et al., 2005) or A.beta.-oligomer related
disease conditions (Lesne et al., 2006; Luibl et al., 2006).
[0003] So far, there is no pharmacotherapy of neurodegenerative
diseases aimed at intervening with the fundamental biological
causes of these diseases. Active or passive immunizaton approaches
targeting disease-associated A.beta. conformers in the case of
Alzheimer disease (Schenk et al., 1999) or shielding the "normal"
substrate conformer PrP.sup.C in the case of prion diseases (White
et al., 2003) have been performed in mouse models of these
diseases. Specifically, administration of mABs in preventing
disease-associated symptoms in mouse models both of prion disease
and Alzheimer disease (Bard et al., 2000; White et al., 2003).
While in the case of anti A.beta. mABs, these seem to pass the
blood brain barrier (BBB) to prevent A.beta. aggregation (Bard et
al., 2000), anti-PrP antibodies for preventing prion disease has
only been successful after peripheral (intraperitoneal) inoculation
when they could act on peripheral sites of replication (Heppner et
al., 2001; White et al., 2003). Thus, while anti-A.beta. mABs seem
to easily pass the BBB, anti-PrP mABs don't.
SUMMARY OF THE INVENTION
[0004] It is therefore one objective of the invention to provide an
antibody or an antibody-like molecule that has improved BBB
permeability and therapeutic options with antibodies for prion
diseases.
[0005] According to the invention an antibody or antibody fragment
is provided which specifically recognizes a prion protein, i.e.
PrP.sup.C and/or PrP.sup.Sc, and which comprises a complementarity
determining region (CDR) according to SEQ ID NO:1, SEQ ID NO:11,
SEQ ID NO:12 and/or SEQ ID NO:13, a retro-inverso D-peptide of said
CDR according to SEQ ID NO:2, and/or an anti-idiotypic antibody or
antibody fragment, which recognizes said CDR, comprising SEQ ID
NO:3. An antibody or antibody fragment according to the invention
that comprises the complementarity determining region 3 of the
heavy chain (CDR3H) alone binds PrP.sup.Sc in a
conformation-specific manner. An antibody or antibody fragment
according to the invention that comprises an M13A or D11R mutant of
CDR3H alone binds PrP.sup.Sc in a conformation-specific manner. An
antibody or antibody fragment according to the invention that
comprises an R10A mutant CDR3H alone binds PrP.sup.C in a
conformation-specific manner. An antibody or antibody fragment
according to the invention that comprises a retro-inverso D-peptide
of CDR3H ((D-)CDR3H) binds specifically to PrP.sup.Sc and exhibits
antiprion activity, demonstrating that these 16 amino
acid-containing peptides, too, have potential as diagnostic and
therapeutic agents in prion diseases.
[0006] An antibody or antibody fragment according to the invention
that comprises an anti-idiotypic antibody or antibody fragment also
exhibits antiprion activity, is able to immunoprecipitate
specifically PrP.sup.Sc and can be used as an immunogen to
circumvent self tolerance to this antigen. All antibodies or
antibody fragments according to the invention bind either PrP.sup.C
and/or PrP.sup.Sc, have improved BBB permeability and offer new
analytic and therapeutic options for prion diseases.
[0007] The term "antibody or antibody fragment", as used herein,
comprises full length antibodies, fragments of antibodies such as
F.sub.ab fragments or scFv, and single regions of antibodies such
as complementarity determining regions (CDRs). However, this term
may also comprise derivatives of said molecules, for example,
retro-inverso peptides of antibody fragments or single CDRs.
[0008] In a preferred embodiment of the invention, the
complementarity determining region (CDR) may be contained in at
least one heavy chain variable region according to SEQ ID NO:4. The
antibody or antibody fragment may further comprise at least one
light chain variable region according to SEQ ID NO:5. In this case,
it is advantageous if at least one heavy chain variable region and
at least one light chain variable region are linked by a linker
peptide, preferably (Gly.sub.4Ser).sub.3. Thus, according to a
preferred embodiment of the invention the antibody fragment is a
scFv fragment comprising at least one heavy chain variable region
according to SEQ ID NO:4 and at least one light chain variable
region according to SEQ ID NO:5. The scFv fragment according to the
invention binds specifically to PrP.sup.C and PrP.sup.Sc and
exhibits antiprion activity so that it can be used in analysis and
therapy of prion-related diseases.
[0009] In order to enhance excretion of a recombinant antibody or
antibody fragment according to the invention, the antibody or
antibody fragment may further comprise at least one signal
sequence, preferably E. coli pelB or a similar leader peptide.
Suitable targeting sequences are also, for example, tissue-specific
or cell-specific antibody fragments that are capable of leading the
antibody or antibody fragment according to the invention to desired
target cells, in particular in brain. Further, signal peptides such
as nuclear localization sequences (NLS) may be fused to the
antibody or antibody fragment according to the invention in order
to guide it within a target cell, for example into the nucleus. If
an antibody or antibody fragment according to the invention further
comprises at least one tag sequence, detection and/or purification
of the antibody or antibody fragment can be facilitated. The tag
sequence may be a c-Myc tag and/or an polyhistidine tag, preferably
hexahistidine. Other tag sequences may be, for example, horse
radish peroxidase, luciferase, or enhanced green fluorescent
protein. That is, the antibody or antibody fragment according to
the invention may be cloned and expressed as a fusion peptide or
protein.
[0010] In a preferred embodiment of the invention, the antibody or
antibody fragment according to the invention comprises the amino
acid sequence according to SEQ ID NO:6, which is a scFv fragment
comprising at least one heavy chain variable region according to
SEQ ID NO:4 and at least one light chain variable region according
to SEQ ID NO:5.
[0011] The invention includes antibodies or antibody fragments
which, on an amino acid level, are at least 85%, preferably 90%,
more preferred 95%, identical to the antibody or antibody fragment
described above. Basically, the invention comprises all L- or
D-peptide derivatives that can compete CDR3H or riCDR3H out of
their interaction with PrPSc, and all L- or D-peptides where
equivalently charged, hydrophobic, aromatic or hydroxyl amino acids
are replaced within each other (positively charged equivalent amino
acids: lysine and arginine, negatively charged equivalent amino
acids: aspartate, glutamate; hydrophobic eqivalent amino acids:
alanine, valine, leucine, isoleucine, methionine; alcoholic
euqivalent amino acids: serine and threonine; neutral equivalent
amino acids: glycine and proline).
[0012] The invention further includes a nucleic acid molecule
selected from a group consisting of [0013] a) nucleic acid
molecules encoding an antibody or antibody fragment comprising the
amino acid sequence according to SEQ ID NO:1; [0014] b) nucleic
acid molecules encoding an antibody or antibody fragment comprising
the amino acid sequence according to SEQ ID NO:2; [0015] c) nucleic
acid molecules encoding an antibody or antibody fragment comprising
the amino acid sequence according to SEQ ID NO:3; [0016] d) nucleic
acid molecules encoding an antibody or antibody fragment comprising
the amino acid sequence according to SEQ ID NO:4; [0017] e) nucleic
acid molecules encoding an antibody or antibody fragment comprising
the amino acid sequence according to SEQ ID NO:5; [0018] f) nucleic
acid molecules encoding an antibody or antibody fragment comprising
the amino acid sequence according to SEQ ID NO:6; [0019] g) nucleic
acid molecules encoding an antibody or antibody fragment comprising
the amino acid sequence according to SEQ ID NO:11; [0020] h)
nucleic acid molecules encoding an antibody or antibody fragment
comprising the amino acid sequence according to SEQ ID NO:12;
[0021] i) nucleic acid molecules encoding an antibody or antibody
fragment comprising the amino acid sequence according to SEQ ID
NO:13; [0022] j) nucleic acid molecules encoding the antibody or
antibody fragment according to the invention; [0023] k) nucleic
acid molecules comprising the nucleotide sequence according to SEQ
ID NO:7; [0024] l) nucleic acid molecules comprising the nucleotide
sequence according to SEQ ID NO:8; [0025] m) nucleic acid molecules
comprising the nucleotide sequence according to SEQ ID NO:9; [0026]
n) nucleic acid molecules comprising the nucleotide sequence
according to SEQ ID NO:10; [0027] o) nucleic acid molecules, the
polynucleotide sequence of which is at least 85%, preferably 90%,
more preferred 95%, identical to the nucleotide sequence of a
nucleic acid molecule of any of a) to n), and which encode an
antibody or antibody fragment that specifically recognizes a prion
protein; [0028] p) nucleic acid molecules, the complementary strand
of which hybridizes to a nucleic acid molecule of any of a) to n),
and which encode an antibody or antibody fragment that specifically
recognizes a prion protein; [0029] q) nucleic acid molecules, the
nucleotide sequence of which differs from the nucleotide sequence
of a nucleic acid molecule of any of a) to p) due to the degeneracy
of the genetic code; and [0030] r) nucleic acid molecules, the
nucleotide sequence of which is complementary to the nucleotide
sequence of a nucleic acid molecule of any of a) to q).
[0031] According to another aspect of the invention, a method for
generating an antibody or antibody fragment that specifically
recognizes a prion protein is provided. The method according to the
invention comprises: [0032] a) generating an antibody that
recognizes a specific domain of the prion protein; [0033] b)
isolating an antigen-specific amino acid sequence from said
antibody; and [0034] c) generating an anti-idiotypic antibody or
antibody fragment, which recognizes said antigen-specific amino
acid sequence.
[0035] This novel method of generating anti-PrP mABs overcomes the
state of prior art and the previous notion that an effective immune
response against PrP was impossible in wild type animals due to
self tolerance. The present invention comprises therefore
immunization with an antibody or antibody fragment binding to an
interaction domain of PrP, which enables a prion protein to
interact with another prion protein, and using it as an immunogen
to circumvent self tolerance to this antigen. Surprisingly, if the
specific domain is an interaction domain of the prion protein, the
anti-idiotypic antibody recognizes PrP.sup.C and/or PrP.sup.Sc and
has antiprion activity. In a preferred embodiment of the method
according to the invention, the antigen-specific amino acid
sequence is a complementarity determining region (CDR), preferably
CDR3H according to SEQ ID NO:1 or a D-peptide retro-inverso
sequence of CDR3H, termed riCDR3H, according to SEQ ID NO:2.
[0036] The invention further relates to a kit comprising the
antibody or antibody fragment according to the invention and/or the
nucleic acid molecule according to the invention.
[0037] The invention also concerns a pharmaceutical preparation
comprising the antibody or antibody fragment according to the
invention and/or the nucleic acid molecule according to the
invention.
[0038] The antibody or antibody fragment according to the invention
or the kit according to the invention or the pharmaceutical
preparation according to the invention can be advantageously used
in diagnosis and/or therapy of prion related diseases or other
diseases where it has been demonstrated that manipulation of the
prion protein by antibody/ligand binding influences the course of
disease. The antibody or antibody fragment according to the
invention or the kit according to the invention or the
pharmaceutical preparation according to the invention can further
be advantageously used for the purpose of eliciting an
immunostimulatory effect.
DETAILED DESCRIPTION OF VARIOUS AND PREFERRED EMBODIMENTS OF THE
INVENTION
[0039] According to the invention, a high-affinity, antiprion
active scFv that could be expressed in high yields as a soluble
protein targeted to the periplasmic space in E. coli is provided.
Due to its reliable antiprion activity, the antibody fragment can
be used for treating prion infections in vivo. The approximately 30
kDa protein is the smallest polypeptide fragment whose antiprion
activity has ever been confirmed by bioassays, next to antiprion
active Fab fragments that are about twice that big (Peretz et al.,
2001). The antibody fragments according to the invention can be
easily modified by recombination, if necessary, for shuttling the
PrP-binding fragment across the BBB, and targeting it to the
subcellular sites of action in the CNS and peripheral sites of
replication.
[0040] Various exemplary and preferred embodiments of the invention
are described below in detail with reference to the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] FIG. 1 shows a schematic drawing of one embodiment of the
antibody fragments according to the invention. The variable heavy
and light chain domains of W226, connected by a flexible
(Gly.sub.4Ser).sub.3-linker domain, (scFv) and W226-Hc were cloned
into the procaryotic expression vector pET22b, allowing the
secretion of the antibody fragments into the periplasm by an
N-terminal pEL-B-leader sequence. In addition C-terminal myc- and
his.sub.6-tags were included. The sequence of the heavy chain CDR3
in L-(CDR3H) or retro inverso-form (riCDR3H) is shown beneath.
[0042] FIG. 2 shows
[0043] a) A Coomassie-stained gel of the purification process
involving metal affinity chromatography (IMAC) and subsequently PrP
affinity chromatography (samples after single purification steps as
indicated). The final fraction after elution from the PrP affinity
column is pure (arrow).
[0044] b) An asymmetric flow field-flow analysis of scFvW226.
Purified scFvW226 consisted of approximately 82% (w/w) monomers and
16% dimers. The largest particles that have been detected were
probably tetramers (.about.120 kDa, .ltoreq.2%).
[0045] c. Quantitation of scFvW226-PrP binding by surface plasmon
resonance spectroscopy (SPR). Recombinant mouse PrP was immobilized
on a CM5-chip. ScFvW226 was injected at concentrations ranging from
x to y at a flow rate of 30 .mu.l/min. Association and dissociation
curves were recorded for 180 s. After each analysis surface was
regenerated with 10 mM NaOH. Curve fitting calculations yielded a
K.sub.D of 2 nM.
[0046] d. A western blot of an immunoprecipitation of normal (N)
and scrapie-infected (Sc) mouse brain homogenates with immobilized
scFvW226. Starting material is blotted on the left panel. Normal
homogenate is pulled down, and the pulled-down material form
scrapie brains is protease-resistant indicating that scFvW226
recognizes both PrP.sup.C and PrP.sup.Sc.
[0047] FIG. 3 shows
[0048] a) A western blot of ScN2a cell lysates treated either by
transfection with recombinant IgG.kappa.-scFvW226 or by transfer
with supernatant from non-infected, IgG.kappa.-scFvW226-transfected
N2a cell conditioned medium. ScN2a cells were either (from left to
right) untreated, transfected with empty vector (pCDNA 3.1.),
transfected with control IgG.kappa.-scFv19B10, or
IgG.kappa.-scFvW226 for 4 days or 7 days. In addition, conditioned
medium of N2a cells transfected with with control
IgG.kappa.-scFv19B10, or IgG.kappa.-scFvW226 was used on ScN2a
cells for 4 days. Furthermore, recombinant control scFv19B10 or
scFvW226 generated in E. coli was used at a concentration of 10 nM
for 7 days. The blot clearly demonstrates that all
scFvW226-containing constructs are antiprion active.
[0049] b) A Western blot of ScN2a cell lysates treated with
recombinant scFvW226 from E. coli. ScN2a cells were treated with
different concentrations of scFvW226 as indicated, for one week.
Subsequently, treatment was discontinued for either one week (upper
panel) or three weeks ("set-off experiment"; lower panel). As
controls, quinacrine (1000 nM) and full length mAB W226 (16 nM)
were used. The blot demonstrates a permanent, dose-dependent,
prion-clearing effect of recombinant scFvW226 in E. coli.
[0050] c) A western blot of ScN2a cell lysates treated with
recombinant scFvW226 from E. coli to determine the minimal
prion-clearing concentration. ScN2a cells were treated with
different concentrations of scFvW226 as indicated for one week. At
a minimal concentration of 4 nM, scFvW226 cleared prions.
[0051] FIG. 4 shows differential conformation-specificity and
antiprion activity of single complementarity determining region 3
of the heavy chain (CDR3H) domain as an L-peptide and as a
retro-inverso D-peptide ((D-)riCDR3H).
[0052] a) A western blot of PK-digested ScN2a cell lysates
untreated (n. t.), treated with 1 .mu.M quinacrine (Q), with
recombinant heavy chain domain (W226-Hc) from E. coli, synthetic
(D-)riCDR3H, or L-CDR3H at the concentrations indicated. Only
(D-)riCDR3H had antiprion activity at 4 .mu.M concentration.
[0053] b) A model of the surface representation of the linearized
CDR3H peptides with the L-peptide at the top and the D-peptide
below. Residues shown in blue and red are basic and acidic,
respectively. It can clearly seen that the positions of the
side-chain functionalities are conserved despite reversal of the
amino acid sequence. Colors for atom positions are light red=O,
light blue=N, gray=C, white=H, yellow=S.
[0054] c) A western blot of pulldown experiments from normal (N) or
scrapie-infected (Sc) mouse brain homogenates with immobilized
L-CDR3H or (D-)riCDR3H. Protease (PK) digestion at 200 .mu.g/mL
indicated the presence of PrP.sup.Sc. Whereas scFvW226 could
precipitate both PrP.sup.C and PrP.sup.Sc (FIG. 2c), the CDR3H
region pulled down only PrP.sup.Sc in a conformation-specfic
manner, as did the (D-)riCDR3H peptide.
[0055] FIG. 5 shows staining of ScN2a cells with
tetramethylrhodamine (TAMRA)-labeled L-CDR3H and (D-)riCDR3H. ScN2a
cells were incubated with 1 .mu.M (D-)riCDR3H-TAMRA (a) or
L-CDR3H-TAMRA (d) for three hours and after washing with PBS, bound
peptides were detected by fluorescence microscopy. To demonstrate
the specificity of binding, ScN2a cells were preincubated with 100
.mu.M scFvW226 30 min before addition of peptides (b and e).
Digestion with trypsin demonstrated protease resistance of
(D-)riCDR3H (c) vs. L-CDR3H (f).
[0056] FIG. 6 shows irradiated splenocytes from B10PL mice that
served as antigen presenting cells (APC), and/or MBP.sub.Ac1-11 T
cell receptor (TCR) transgenic (Tg) CD4.sup.+ T cells were treated
with scFvW226 or mAB W226 and antigen (Ag). T cell proliferation
was measured by .sup.3[H] thymidine incorporation. APC and/or
Ag-specific T cells were treated with (A) a single chain Fv
fragment of W226 (scFv), or full length mAB W226 (B), or isotype
controls (ISO). Targeting PrP.sup.c with any of these molecules
resulted in the augmented proliferation of Ag-specific T cells.
[0057] FIG. 7 shows a Western blot of an immunoprecipitation
experiment where a relative specificity for W226 to sheep prions,
but not mouse prions can be seen. The two leftmost lanes depict the
input sheep homogenate material (N=normal; Sc=scrapie) used for
immunopreciptiation, then (from left to right) a control without
antibody, and increasing concentrations of antibody (AB) as
depicted. The two righmost lanes are from mouse as a comparison. It
can be seen that only high concentrations of W226 during the
immunoprecipitation result in weak binding of PrP.sup.C from normal
sheep homogenate whereas it pulls down readily PrP.sup.C from mouse
brain homogenates.
EXAMPLES
[0058] scFvW226 Construction, Expression, and Characterization
[0059] The original mAB W226 was dervied from a hybridoma cell line
generated after immunization with purified mouse PrP.sup.Sc For the
IgG1 subtype mAB, a monovalent dissociation constant (K.sub.D) with
recombinant PrP was determined to be 0.5 nM (by surface plasmon
resonance [SPR]) and, by immnoprecipitation, binding to both
PrP.sup.C and PrP.sup.Sc was detected.
[0060] The variable light and heavy chains were cloned as a scFv
into the pelB containing pET22b vector, including a
(Gly.sub.4Ser).sub.3-spacer (Huston et al., 1988) between H and L
chain, and a C-terminal cmyc and His6 tag (see FIG. 1). The pelB
leader sequence would target the protein to the bacteria's
periplasmic space and generate a correctly folded and soluble
protein.
[0061] Under optimized conditions (see Materials and Methods), the
expression yield was 10 mg soluble protein per liter of bacterial
culture that were subsequently purified by IMAC (NiNTA, Qiagen,
Germany) and affinity purification (sepharose column crosslinked
with recombinant mouse PrP; FIG. 2a). The Far-UV circular dichroism
spectrum of scFvW22 contained, like many scFvs (Pledger et al.,
1999), mostly .beta.-structure: a secondary structure estimate
yielded 6% .alpha.-helix, 45% .beta.-sheet, 11% .beta.-turn and 39%
unfolded structure (Lobley et al., 2002). Purified scFvW226 was
mainly monomeric (85%, FIG. 2b) and the K.sub.D measured by SPR
with recombinant mouse PrP was determined to be 2 nM, i.e. only
four times lower than for the full length mAB (FIG. 2c). ScFvW226
retained binding activity to recombinant mouse PrP after incubation
at 60.degree. C. or in 90% serum at 37.degree. C. for 72 h,
indicating a high stability under physiological conditions.
ScFvW226 retained the binding characteristics from its full length
ancestor in that it immunopreciptiated both PrP.sup.C and
PrP.sup.Sc from brain homogenates (FIG. 2d).
[0062] The epitope of scFvW226 was mapped to comprise the linear
polypeptide sequence WEDRYYREN (residues 145-153) in helix 1 of PrP
using a PepSpot library (Jerini Peptide Technology, Germany).
[0063] scFvW226 Antiprion Activity
[0064] Next, scFvW226 was probed for antiprion activity. When
scFvW226 cloned behind the IgG.sub..kappa.-signal sequence was
transfected for secretion into ScN2a cells a clear time-dependent
antiprion effect was observed (FIG. 3a; compare 4 days after
transfection with 7 days after transfection). Similarly, when
scFvW226 was expressed in non-infected N2a cells and the
supernatant of the conditioned medium was transfered to
untransfected ScN2a cells after four days, prions were cleared
(FIG. 3a). A control scFv derived from an antibody recognizing only
a subpopulation of PrP to be antiprion active.
[0065] When ScN2a cells were treated with purified scFvW226
generated in E. coli, prion-clearing effects within concentrations
>3.2 nM (FIG. 3b) was observed. These antiprion effects were
permanent since three weeks after discontinuation of scFvW226
administration, no PK-resistant immunoreactivity corresponding to
PrP.sup.Sc reappeared (FIG. 3b, lower panel). The smallest
effective concentration clearing prions from ScN2a cells as seen in
Western blots was narrowed down to 4 nM (FIG. 3c).
[0066] When lysates of ScN2a cells treated with scFv for either ten
days or three weeks were inoculated into tg20 indicator mice,
scFvW226 concentrations as low as 10 nM were demonstrated to
abolish prions from ScN2a cells (Table 1), thus confirming the
results from the ScN2a cells.
[0067] Miniaturization of scFvW226
[0068] Smaller fragments than the combined CDR regions from heavy
or light chain domains have sometimes shown full biological
activity (Bourgeois et al., 1998; Colby et al., 2004; Jackson et
al., 1999; Kim et al., 2006). For .alpha.-PrP antibodies, the heavy
chain of mAB 6H4 has been shown to be sufficient for antiprion
activity in vivo (Heppner et al., 2001). Cloning and expression of
only the heavy chain variable domain of scFvW226 containing all
three CDRs in E. coli, and administration to ScN2a cells failed to
clear prions at concentrations where the entire scFvW226 would
(FIG. 4a). When each of the CDRs were expressed as recombinant,
cmyc- and His-tagged proteins in E. coli, the third CDR of the
heavy chain domain (CDR3H), as well as the light chain exhibited
weak binding to recombinant mouse PrP by an enzyme-linked
immunosorbent assay.
TABLE-US-00001 TABLE 1 Bioassays of treated ScN2a cells in tg20
mice number dosage of treatment number incubation of ScN2a fragment
time of sick time construct cells (.times.10.sup.5) [nM] [days]
n/n.sub.0 [days] PBS 0.8 -- 10 5/5 130 .+-. 20 scW226 0.8 10 10 0/5
scW226 0.8 30 10 0/5 scFvW226 0.8 100 10 0/5 scFvW226 0.8 300 10
0/5 PBS 2.8 -- 21 d 5/5 75 .+-. 2 scFvW226 2.8 300 21 d 0/5 W226-Hc
2.8 300 21 d 5/5 78 .+-. 3 ScN2a cells were treated with scFv in
different concentrations, and for different times; even at the
lowest concentration (10 nM) scFvW226 cleared prions completely.
PBS treatment (control) or treatment with 320 nM W226-Hc did not
clear prions.
[0069] CDR3H is the most variable region among all CDRs (Shirai et
al., 1996). In many antibodies, this region alone has been shown to
exhibit weak binding to the epitope (Feng et al., 1998; Heap et
al., 2005; Monnet et al., 1999). When the CDR3 heavy chain (CDR3H)
was expressed in E. coli with a cmyc and His.sub.6 tag or
synthesized without the tags and administered to ScN2a cells, it
exhibited no antiprion activity (FIG. 4a).
[0070] A Retro-Inverso (D-) Peptide of CDR3H is Antiprion
Active
[0071] Retro-inverso D-peptide analogues of corresponding
L-peptides are D-peptides ("inverso") in the reverse sequence order
("retro"), attempting to mimick the side chain topology of the
L-peptide while having a different backbone with resistance to
proteolysis by L-proteases in vivo. Binding of these peptides to
the antigen would be predicted to occur only when the majority of
binding interactions stems from side chain interactions rather than
involving polypeptide backbone interactions. D-peptides offer
advantages over L-peptides in that they have a dramatically
increased half life time in vivo (Briand et al., 1995; Guichard et
al., 1994; Levi et al., 2000). Surprisingly, the (D-)riCDR3H
exhibited antiprion activity at concentrations of 4 .mu.M where
CDR3H had no activity (FIG. 4a). Attempts to measure different
affinities of the peptides to PrP by SPR were limited due to the
small molecular size of the peptides; the K.sub.Ds of both peptides
has been estimated to be in the range of 1-10 .mu.M, and no huge
differences in the binding of the L- and the D-peptide could be
observed.
[0072] For further characterizing the differences that would be
associated with differential antiprion activity of (D-)riCDR3H as
opposed to CDR3H, live immunofluorescence stainings of ScN2a cells
with TAMRA-labeled riCDR3H and CDR3H have been performed (FIG. 5).
While riCDR3H exhibited staining all over the cell, CDR3H stained
only intracellular compartments, likely after its endocytosis. Both
stainings could be competed by scFvW226 indicating that both
riCDR3H and CDR3H bound to the same antigens, i.e. PrP. As
expected, only CDR3H could be digested by trypsin (FIG. 5). These
findings indicated that the differential antiprion activity of
riCDR3H was likely due to different in vivo characteristics that
included half life time and subcellular targeting.
[0073] CDR3H and (D-)riCDR3H are Conformation-Specific Ligands fo
PrP.sup.Sc
[0074] To investigate whether the CDR peptides had maintained PrP
binding characteristics, pull down experiments with
sepharose-immobilized peptides of brain homogenates from normal and
RML-infected mice were performed. Surprisingly, CDR3H and
(D-)riCDR3H pulled down only PrP from scrapie-infected homogenates
that after digestion with PK revealed partial protease resistance
indicating that this conformer was PrP.sup.Sc (FIG. 4c). Thus,
compared to scFvW226, CDR3H peptides changed binding specificity
and acquired conformation-specific binding to PrP.sup.Sc, and this
binding seemed side-chain mediated since both CDR3H and (D-)riCDR3H
bound PrP.sup.Sc, although the L-peptide a little stronger (FIG.
4c). The conformation-specificity of CDR3H and (D-)riCDR3H for
PrP.sup.Sc could explain the difficulties of determining K.sub.Ds
with recombinant mouse PrP which is though to resemble PrP.sup.C
rather than PrP.sup.Sc.
[0075] Use of scFvW226 or its Derivatives for Strain Specific
Detection of Prions
[0076] In FIG. 7 it is shown that not all prions (PrP.sup.Sc) are
recognized equally well by the W226 ligand. This characteristic may
now be used to distinguish strains. For example, by using W226
traditional classic sheep scrapie may be distinguished from BSE
scrapie, i.e. prions that originate from cattle infected with
prions and transmitted to sheep. These prions are particularly
dangerous for humans. Antibody fragments may be mutagenized at
single amino acid residues to increase strain-specific
recognition.
[0077] Use of scFvW226 or its Derivatives for Design and
Construction of Fusion Proteins with Improved Diagnostics and
Therapeutic Activity
[0078] Using either the PrP.sup.C and PrP.sup.Sc-recognizing
scFvW226 or the PrP.sup.Sc-specific CDR3H, fusion proteins can be
made that facilitate diagnostics: horse radish peroxidase can be
recombinantly fused to the gene of these antibody fragments and be
used to detect prions enzymatically in a one-step reaction.
Equally, these antibody fragments can be fused to enhanced
fluorescent protein (EFP) or luciferase to directly attach a label.
It may also be of advantage to add additional signal sequences to
these antibody fragments allowing either improved passage through
the blood-brain barrier and/or arrival at particular CNS
structures. It is also conceivable to add signal sequences or cell
transduction sequences allowing the recombinant antibody fragment
to reach defined cellular compartments. The antibody fragments may
also be recombinantly combined with each other to yield antibody
fragements of variable size capable of binding PrP at multiple
sites. This may lead to decreased dissociation of the antibody
fragment from PrP due to increased avidity. These constructs may be
particularly useful for immunization experiments. It is also
conceivable to combine scFvW226-derived antibody fragments with
other recombinant antibodies or protein ligands by construction
respective fusion proteins; these may be advantageous since they
combine the bioavailability characteristics of both fragments.
[0079] Anti-Idiotypic Antibodies to scFvW226 (CDR3H) Bind PrP
[0080] The epitopes of full length mAB W226 and scFvW226 were
mapped to helix 1 of the prion protein (residues WEDRYYREN). Since
helix 1 is known to be an interaction site in the
PrP.sup.C/PrP.sup.Sc complex (Solforosi et al., 2007), the minimal
PrP-binding domain within scFvW226, the complementarity-determining
region (CDR) 3 of the heavy chain (CDR3H) was used in an
immunization experiment in order to generate anti-idiotypic
antibodies to PrP. These anti-CDR3H antibodies should ultimately
resemble the PrP helix 1 domain and therefore be able to bind to
PrP.
[0081] Wild type 129 SvEv mice have been immunized with
recombinantly expressed CDR3H or synthesized riCDR3H crosslinked to
KLH with Linaris adjuvant (in both cases a total of 3 boosters over
10 weeks), and generated hybridoma of their spleens by standard
methods. For the mouse immunized with CDR3H, 28 hybridoma secreting
monoclonal antibodies (mABs) recognizing both the immunogen and
mouse PrP were generated; 8 clones were generated that only
recognized the immunogen. The anti-PrP mABs had different
characteristics in that by immunoprecipitation they recognized
PrP.sup.C and PrP.sup.Sc, only PrP.sup.C or only PrP.sup.Sc. One
clone from this fusion is mAB 7A7. This antibody was able to
immunoprecipitate specifically PrP.sup.Sc.
[0082] This novel method of generating anti-PrP mABs overcame the
state of prior art and the previous notion that an effective immune
response against PrP was impossible in wild type animals due to
self tolerance. The present invention comprises therefore
immunization with an antibody or antibody fragment binding to an
interaction domain of PrP and using it as an immunogen to
circumvent self tolerance to this antigen.
[0083] Immunizing a mouse with chemically synthesized riCDR3H
crosslinked to keyhole limpet hemocyanin (KLH) by a similar
protocol as described above and fusing the spleen to generate
hybridoma by standard methods, equally resulted in anti-PrP
antibodies of differential conformation specificity. Thus, the
riCDR3H is able to overcome self tolerance of humoral immune
response against PrP.
[0084] In order to demonstrate that this active immunization
strategy could be used to protect from prion infection, five CD1
mice were immunized four times in 2 week intervals with CDR3H
peptide produced in and purified from E. coli, with the first two
immunizations utilizing ABM-ZK adjuvant (Linaris, Germany) and the
last two boosts with ABM-N (Linaris, Germany) as adjuvant. These
five mice as well as five non-immunized controls were then
inoculated intraperitoneally with a 10-4 dilution of a proven 10%
homogenate scrapie (RML) infected terminally sick mouse brain in
PBS (20 micL/mouse). Control mice died with an average incubation
time of 195 days. From the immunized mice, one mouse survived
(>10 months), whereas the other four died with an average
incubation time of 205 days. Thus, CDR3H is able to protect from
prion disease. These immunization procedures can be further
improved by providing immunogens where several CDR3H fragments are
cloned behind each other resulting in double, triple, or multiple
identical sites of the immunogen in one protein. These repetitive
structures may particularly well be recognized by the immune
processing machinery and lead to an increased anti-PrP immune
response. Similarly, riCDR3H can be chemically crosslinked to a
D-peptide scaffold to result in a multiple antigenic peptide (MAP).
This MAP equally presents a repetitive structure favoring a strong
anti-PrP immune response.
[0085] Antiprion Activity of scFvW226 in Prion-Infected Mice
[0086] In order to demonstrate therapeutic effects of a passive
immunization strategy with scFvW226 against ongoing prion disease,
an experiment was set up where C57/B6 mice were inoculated
intraperitoneally with a 10-3 dilution of a proven 10% homogenate
scrapie (RML) infected terminally sick mouse brain in PBS (20
micL/mouse). Thirty days after inoculation, intraperitoneal
treatment with either full length purified mABW226 or scFvW226 was
started. Nine untreated controls, and five mice treated with either
mABW226 2 mg/mouse or 1 mg scFvW226/mouse twice a week for an
unlimited period. There had been a steady loss of weight for the
control mice prior to scrapie sickness, whereas none of the treated
mice experienced any weight loss. There was a significant effect on
increasing the survival time by administration of scFvW226 or full
length antibody. This experiment proved that scFvW226 (or mABW226)
was able to prevent prion disease when given after inoculation.
[0087] Immunostimulatory Effect of mAB W226 and scFvW226
[0088] To investigate whether scFv W226 or full length mAB W226 had
effects on the T cell immune response, T cells were isolated from a
transgenic mouse expressing T cell receptor specific for myelin
basic protein (MBP). These T cells would specifically proliferate
upon encountering MBP 1-11 antigen, a peptide comprising the
N-terminal first eleven residues of MBP. It could be demonstrated
that addition of scFvW226 (A.) or mAB W226 (B.) increased T cell
proliferation in the presence of MBP 1-11 indicating that scFvW226
or mAB W226 have immunostimulating effects.
[0089] Thus, the potential application of scFvW226 or its
derivatives is also to counteract immunosuppressed conditions like
aplastic anemia, leukemias, or HIV/AIDS, or others, or as
immunoadjuvant when the immune response in individuals is weakened,
for example in older patients receiving active immunizations.
TABLE-US-00002 TABLE 2 Mutagenesis of CDR3H No. Peptide Sequence
Specificity Affinity 1. LH3 YFCARWNWERDAMDYWG PrP.sup.Sc 0 2. LH3A3
YFAARWNWERDAMDYWG PrP.sup.Sc 3. LH3A5 YFCAAWNWERDAMDYWG PrP.sup.Sc
4. LH3A6 YFCARANWERDAMDYWG PrP.sup.Sc 5. LH3A7 YFCARWAWERDAMDYWG
PrP.sup.Sc (+) 6. LH3A8 YFCARWNAERDAMDYWG PrP.sup.Sc 7. LH3A9
YFCARWNWARDAMDYWG PrP.sup.Sc 8. LH3A10 YFCARWNWEADAMDYWG PrP.sup.C
9. LH3A11 YFCARWNWERAAMDYWG PrP.sup.Sc 10. LH3A13 YFCARWNWERDAADYWG
PrP.sup.Sc + 11. LH3A14 YFCARWNWERDAMAYWG PrP.sup.Sc (+) 12. LH3A15
YFCARWNWERDAMDAWG PrP.sup.Sc 13. LH3D5 YFCADWNWERDAMDYWG PrP.sup.Sc
(-) 14. LH3R9 YFCARWNWRRDAMDYWG PrP.sup.Sc (+) 15. LH3D10
YFCARWNWEDDAMDYWG PrP.sup.Sc (+) 16. LH3R11 YFCARWNWERRAMDYWG
PrP.sup.Sc + 17. LH3R14 YFCARWNWERDAMRYWG PrP.sup.Sc In the
peptides, for each subsequent residue, the amino acid was
substituted with an alanine, that is thought to abrogate side chain
interactions but maintain secondary structure (Nos. 1-12).
Evaluation was done by comparing the specificity and affinity for
PrPSc pulldown to that of peptide LH3 (CDR3H).
[0090] Mutagenesis of CDR3H
[0091] Table 2 shows a mutagenesis experiment of peptides derived
from SEQ ID NO:1 (CDR3H, here termed LH3). In the derived peptides,
for each subsequent residue, the amino acid was substituted with an
alanine (Nos. 1-12), that is thought to abrogate side chain
interactions but maintain secondary structure. Further
substitutions have also been accomplished as shown in Table 2 (Nos.
13-17). Evaluation was done by comparing the specificity and
affinity for PrP.sup.Sc pulldown to that of peptide LH3 (CDR3H, SEQ
ID NO:1). There are three remarkable results that serve to further
narrow the role of side chains of the LH3 peptide in PrP.sup.Sc
(prion-) specificity:
[0092] 1. Mutagenesis of R10 to A10 (No. 8: LH3A10, SEQ ID NO:13)
abrogates PrP.sup.Sc specificity: this peptide binds to
PrP.sup.C.
[0093] 2. The two changes M13A (No. 10: LH3A13, SEQ ID NO:11) and
D11R (No. 16: LH3R11, SEQ ID NO:12) increase affinity for
PrP.sup.Sc.
[0094] 3. Changes at residues N7A (No. 5: LH3A7), D14A (No. 11:
LH3A14), E9R (No. 14: LH3R9), R10D (No. 15: LH3D10) slighlty
increase affinity for PrP.sup.Sc whereas change R5D (No. 13: LH3D5)
slighlty decreases affinity for PrP.sup.Sc.
[0095] Exemplary Materials and Methods
[0096] Constructs
[0097] W226 hybridoma secreting IgG1 mAB and recognizing both
PrP.sup.C and PrP.sup.Sc had been generated by standard fusion
procedure of myleoma cells with splenocytes from a PrP knockout
mouse (Bueler et al., 1992) immunized with purified mouse
PrP.sup.Sc To prepare a single chain Fv construct (scFv), mRNA
purified from W226 hybridoma was used for PCR amplification with
the following primer set: 5'-AAAACCATGGCGGAGGTCCAGCTGCAGCAGTC 3'
(V.sub.H forward) and
5'-TTTTGCCGGCCAGTGGATAGTCAGATGGGGGTGTCGTTTTGGC-3' (V.sub.H reverse)
or 5'-AAAGGATCCGACATTGTGATGACCCAGTCT-3 (V.sub.L forward) and
5'-AAAAGCGGCCGCGGATACAGTTGGTGCAGCATC-3' (V.sub.L reverse). PCR
products were digested with NgoMIV (V.sub.H) or BamHI (V.sub.L) and
ligated to the NgoMIV and BamHI site of a linker oligonucleotide
coding for a (Gly.sub.4Ser).sub.3 linker domain (Huston et al.,
1988). An 800 bp fragment corresponding to the correct ligation
product was eluted from an agarose gel and amplified using the
V.sub.H forward and V.sub.L reverse primer. The product was cut
with NcoI and EagI and ligated into the procaryotic expression
vector pET22b (Novagen), allowing the expression with an N-terminal
pelB leader sequence and a c-terminal His.sub.6-tag (see FIG. 1).
In addition a c-myc-tag was cloned into the EagI/XhoI sites between
the scFv and the His.sub.6-tag. For construction of only the heavy
chain domain (W226-Hc), V.sub.H was amplified with appropriate
primers allowing the cloning via NcoI and EagI into
pET22b-Myc/His.sub.6. For eukaryotic expression of scFvW226, the
combined scFvW226 cDNA was amplified with a 5'-primer including
including a IgG.kappa.-leader sequence (Donofrio et al., 2005) and
ligated via HindIII/EcoRI (W226) into pCDNA3.1 (Invitrogen).
[0098] Peptides
[0099] CDR3H corresponding to the sequence
NH2-YFCARWNWERDAMDYWG-COOH (one letter amino acid code, SEQ ID
NO:1) and the retro-inverso D-peptide [(D-)riCDR3H] corresponding
to the sequence NH2-gwydmadrewnwracfy-COOH (one letter amino acid
code, small letter convention for D-peptides, SEQ ID NO:2) were
synthesized either unlabeled or N-terminally linked to
6-Carboxy-tetramethylrhodamine (TAMRA) by JPT Peptide Technology
(Berlin, Germany).
[0100] Protein Expression and Purification
[0101] Expression of scFvW226 or W226-Hc was induced in BL21
(.lamda.DE3) Rosetta (EMD, Novagen Brand, Madison, Wis.): bacteria
were grown at 37.degree. C. to high density (OD.sub.600>2.0) in
a 2 L-fermenter (MoBiTec, Gottingen, Germany) and cooled down on
ice before induction with 0.5 mM IPTG at 15.degree. C. over night.
Cell pellets were lyzed in 20 mM Tris pH 8.0, 1% T-X100, 500 mM
NaCl, 5 mM imidazole, 20 mM MgCl.sub.2, 1 mM PMSF, 1 mg/ml lysozyme
and 500U DNase. Lysates were cleared by centrifugation and soluble
protein in the supernatant was purified via Ni-NTA columns (Qiagen,
Hilden, Germany). After loading, the column was washed with 10
column volumes (CV) 20 mM Tris pH8.0, 500 mM NaCl, 1% TX-100, 5 mM
imidazole, 10 CV 20 mM Tris pH8.0, 500 mM NaCl, 1% TX-100, 20 mM
imidazole and 10 CV 20 mM Tris pH 8.0, 1000 mM NaCl, 5 mM
imidazole. Bound proteins were eluted with 20 mM Tris pH 8.0, 300
mM NaCl, 300 mM imidazole yielding a purity of about 60% for
scFvW226 and 90% for W226-Hc. Eluted scFvW226 was further purified
to >95% purity by affinity chromatography employing recombinant
mouse PrP (Korth et al., 1999) coupled to NHS-Sepharose (Amersham)
according to manufacturer's recommendations. From the affinity
column, scFvW226 was eluted with 100 mM glycine pH 2.5 and
immediately neutralized with 100 mM Tris pH 8.8. Finally, purified
antibody fragments were dialyzed twice against PBS. The mass of
scFvW226 was measured by mass pectromtery and found to be identical
to the calculated one.
[0102] Pull-Down Experiments
[0103] scFvW226, W226-Hc, riCDR3H and CDR3H were coupled to
NHS-sepharose. 10% mouse brain homogenates prepared from C57BL/6 or
RML-infected C57BL/6 mice (Chandler, 1961) were diluted 1:10 in in
20 mM Tris HCl pH 8.0, 150 mM NaCl, 0.3% sarcosyl and precleared by
centrifugation for 15 min at 22.000.times.g. 1 mL thereof was
incubated with 20 .mu.L of loaded beads at 4.degree. C. over night.
In a positive control experiment, 5 .mu.g of recombinant mouse PrP
in the same buffer was used. After incubation, beads were washed
twice in IP1-buffer (50 mM Tris pH7.5, 150 mM NaCl, 1% NP40, 0.5%
DOC), IP2-buffer (50 mM Tris pH 7.5, 500 mM NaCl, 0.1% NP40, 0.05%
DOC) and IP3-buffer (50 mM Tris pH7.5, 0.1% NP40, 0.05% DOC). Where
necessary, beads were also incubated with 4 .mu.g Proteinase K
(Merck, Darmstadt, Germany) in 20 .mu.L IP3 buffer prior elution of
bound PrP with 2.times. loading buffer at 95.degree. C. The eluates
were separated on a 4%-20%-Tris HCl gel (Biorad, USA) and PrP was
detected by Western Blot using mAb W226.
[0104] Circular Dichroism (CD) Analyses
[0105] Far-UV CD spectra (195-250 nm) were recorded using a Jasco
J-810 spectrometer. Sample conditions: 3 .mu.M protein in 20 mM
NaPO.sub.4 pH 7.5, 0.2 mM EDTA at room temperature (2 mm cuvette).
Scan conditions: 20 nm/min scan speed, 100 mdeg sensitivity, 0.2 nm
pitch, 1 nm band width, 2 s response time, 40 accumulations.
[0106] Asymmetric Field-Flow Fractionation (aFFF)
[0107] System: Eclipse 2 equipped with HELEOS, Optilab Rex (Wyatt
Technologies, USA) and a multiple wavelength detector (Agilent,
USA). Software: Eclispe 2.5 and Astra 5.3.1.4. Conditions: scFvW226
was separated in 10 mM Tris-HCl pH 8, 50 mM NaCl, 1 mM EDTA with a
1 ml/min channel flow, using a 490 .mu.m spacer and 5 kDa MWCO
cellulose membrane. Flow scheme: sample inject (50 .mu.L/75
.mu.g).fwdarw.focussing (2 min, 3 mL/min cross-flow
(V.sub.x)).fwdarw.1.sup.st elution phase (20 min, 2 mL/min linear
V.sub.x).fwdarw.2.sup.nd phase (5 min 2.0-0.15 mL/min V.sub.x
gradient).fwdarw.3.sup.rd phase (5 min V.sub.x off).
[0108] Surface Plasmon Resonance Analysis (SPR)
[0109] Binding kinetics were determined on a Biacore 1000 (Biacore
AB, Uppsala, Sweden). Recombinant mouse PrP (1 .mu.M) was diluted
in 10 mM NaOAc pH 4.5 and immobilized on a EDC/NHS activated
CM5-chip (Biacore) at 5 .mu.l/min. After immobilization and
blocking with ethanolamine, the chip was washed with 50 mM NaOH
until a steady signal was obtained. Final surface density was about
2000 RU. All kinetic SPR analysis were run at 5 .mu.l/min PBS flow
and antibody fragments were injected at different concentrations
ranging from x to y nM. Association and dissociation was recorded
for 180 s respectively. After each cycle, the surface was
regenerated with a x s pulse of 50 mM NaOH. Kinetic data were
calculated using BIAevaluation 4.1 software according to a 1:1
(Langmuir) binding model.
[0110] PrP.sup.Sc Inhibition Assay
[0111] Inhibition by Purified Antibody Fragments
[0112] ScN2a cells (Bosque and Prusiner, 2000; Butler et al., 1988)
were grown in MEM, supplemented with 2 mM L-glutamine, 100 U/mL
penicillin, 100 .mu.g/mL streptomycin and 10% FCS. For treatment,
ScN2a cells were seeded in 60 mm dishes and incubated with antibody
fragments for 7 days. After 3 days, medium/antibody fragments were
changed. Cells were lyzed in 500 .mu.L lysis buffer (50 mM Tris HCl
pH8.0, 150 mM NaCl, 0.5% T-X100, 0.5% DOC) and equal amounts of
lysates were treated with Proteinase K (20 .mu.g/mL) for 30 min at
37.degree. C. After stopping protease digestion with 100 .mu.M
PMSF, PrP.sup.Sc in 400 .mu.L lysis buffer was pelleted at
100.000.times.g in a TLA-55 rotor in a n Optima ultracentrifuge
(BeckmanCoulter, USA). PrP.sup.Sc was detected after separation on
a 4% -20% Tri-HCl gel (Biorad, USA) by Western Blot using mAb
W226.
[0113] Inhibition by Antibody Fragments Expressed in Cells
[0114] ScN2a cells were splitted in 60 mm dishes the day before
transfection to obtain 50% confluency and 1.3 .mu.g pcDNA plasmid
encoding scFvW226 or control scFv was transfected with HiPerfect
(Qiagen, Germany) according to manufacturer's instructions. After
four days cells either were lyzed and analyzed for PrP.sup.Sc as
described above or they were transferred to a 100 mm dish and
incubated for additional 3 days before lysis. In addition,
non-infected N2a cells were transfected in the described way and,
after four days, conditioned medium was transferred to freshly
seeded ScN2a cells, which subsequently were incubated for another
four days.
[0115] Bioassay
[0116] Two separate treatment experiments of determining the
presence of prions after scFvW226 or full length mAB W226 treatment
by inoculation in tg20 mice (Fischer et al., 1996) were performed:
ScN2a cells were grown in 60 mm dishes and treated with 10 nM, 30
nM, 100 nM or 300 nM for 10 days with two splittings and scFvW226
renewals. In a second experiment, ScN2a cells grown in 60 mm dishes
and treated with either 320 nM W226-scFv or W226-Hc. After three
weeks of treatment, including two passages, cells were collected by
scraping, washed in PBS, counted and resuspended in 100 .mu.l PBS,
followed by five cycles of freeze/thawing. For both experiments, 20
.mu.l of lysates corresponding to 0.8 or 2.8.times.10e5 cells were
injected i.c. into five tg20 mice for each treatment condition.
[0117] ScN2a Cell Immunofluorescence Staining
[0118] Live ScN2a cells were washed with PBS and, in one condition,
preincubated with medium containing 100 .mu.M scFvW226 for 30 min
at RT. Subsequently, 1 .mu.M of undigested or trypsin-digested
TAMRA-labeled riCDR3H or CDR3H was added. Trypsin-digestion was
carried out with 100 .mu.g trypsin for 3 h at 37.degree. C. After
incubation with labeled peptides of 3 h, cells were fixed with 4%
paraformaldehyde and washed three times with PBS before
inspection.
[0119] The prion protein, PrP, exists in several stable
conformations, with the presence of one conformation, PrP.sup.Sc,
associated to transmissible neurodegenerative diseases. Targeting
PrP by high-affinity ligands has been proven an effective way of
preventing peripheral prion infections. Here, recombinant single
chain fragments of the variable domains (scFv) of a monoclonal
antibody have been generated in E. coli, originally raised against
purified PrP.sup.Sc and recognizing both PrP.sup.C and PrP.sup.Sc.
This scFv fragment had a dissociation constant (K.sub.D) with
recombinant PrP of 2 nM and cleared prions in ScN2a cells at 4 nM,
as demonstrated by bioassay. Recombinant expression of only its
complementarity determining region 3 of the heavy chain (CDR3H) led
to conformation-specific recognition of only PrP.sup.Sc in
solution, however, antiprion activity was lost. Synthesis of a
retro-inverso D-peptide of CDR3H reinstated antiprion activity.
Thus, 1. scFvW226 is so far the smallest polypeptide with bioassay
proven antiprion activity and 2. differential
conformation-specificity can be regulated by orchestrating the
participation of different CDRs.
[0120] Mutagenesis Experiments
[0121] Peptides were synthesized by JPT Peptides (Berlin, Germany)
at >70% purity and HPLC purified. Peptides were coupled to NHS
sepharose via their free N-terminal amine and used in a pull-down
experiment as described above. Evaluation was then done by
comparing the specificity and affinity for PrPSc pulldown to
peptide CDR3H (also termed LH3). The results of this investigation
are listed in Table 2.
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Sequence CWU 1
1
13117PRTMus musculus 1Tyr Phe Cys Ala Arg Trp Asn Trp Glu Arg Asp
Ala Met Asp Tyr Trp1 5 10 15Gly217PRTArtificialRetro-inverso
D-peptide of CDR3H [(D-)riCDR3H] 2Gly Trp Tyr Asp Met Ala Asp Arg
Glu Trp Asn Trp Arg Ala Cys Phe1 5 10 15Tyr3268PRTMus musculus 3Met
Ala Asp Val Lys Leu Gln Glu Ser Gly Ala Glu Leu Val Lys Pro1 5 10
15Gly Ala Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
20 25 30Ser Tyr Tyr Met Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
Glu 35 40 45Trp Ile Gly Glu Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe
Asn Glu 50 55 60Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser
Ser Ser Thr65 70 75 80Ala Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu
Asp Ser Ala Val Tyr 85 90 95Tyr Cys Thr Arg Asp Tyr Arg Tyr Ala Trp
Phe Ala Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ala
Glu Ser Gln Ser Phe Pro Asn Val 115 120 125Phe Pro Leu Ala Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 130 135 140Gly Gly Ser Asp
Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val145 150 155 160Ser
Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile 165 170
175Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro
180 185 190Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg
Phe Ser 195 200 205Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr 210 215 220Leu Lys Ile Ser Arg Val Glu Ala Glu Asp
Leu Gly Val Tyr Tyr Cys225 230 235 240Phe Gln Gly Ser His Val Pro
Tyr Thr Phe Gly Gly Gly Thr Lys Leu 245 250 255Glu Ile Lys Arg Ala
Asp Ala Ala Pro Thr Val Ser 260 2654179PRTMus musculus 4Met Lys Tyr
Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln
Pro Ala Met Ala Met Ala Glu Val Gln Leu Gln Gln Ser Gly 20 25 30Ala
Glu Leu Ala Arg Pro Gly Thr Ser Val Asn Leu Ser Cys Lys Thr 35 40
45Ser Gly Tyr Ser Phe Thr Gly Tyr Gly Val Ser Trp Val Lys Gln Arg
50 55 60Ile Gly Gln Gly Leu Glu Trp Ile Gly Glu Ile Tyr Pro Arg Ser
Gly65 70 75 80Asn Thr Tyr Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr
Leu Thr Ala 85 90 95Asp Lys Ser Ser Asn Thr Ala Tyr Met Glu Leu Arg
Ser Leu Thr Ser 100 105 110Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg
Trp Asn Trp Glu Arg Asp 115 120 125Ala Met Asp Tyr Trp Gly Gln Gly
Thr Ser Val Thr Val Ser Ser Ala 130 135 140Lys Thr Thr Pro Pro Ser
Asp Tyr Pro Leu Ala Gly Gly Gly Gly Ser145 150 155 160Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln 165 170 175Thr
Pro Leu 5123PRTMus musculus 5Asp Ile Val Met Thr Gln Thr Pro Leu
Ser Leu Pro Val Ser Leu Gly1 5 10 15Asp Gln Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Glu Ser Ser 20 25 30Ser Gly Tyr Pro His Leu Asn
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45Pro Gln Leu Leu Ile Tyr
Arg Val Ser Asn Arg Phe Ser Gly Val Leu 50 55 60Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Gln Ile65 70 75 80Ser Arg Val
Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Leu Gln Ile 85 90 95Thr His
Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
110Arg Ala Asp Ala Ala Pro Thr Val Ser Ala Ala 115
1206311PRTArtificialscFv 6Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala
Gly Leu Leu Leu Leu Ala1 5 10 15Ala Gln Pro Ala Met Ala Met Ala Glu
Val Gln Leu Gln Gln Ser Gly 20 25 30Ala Glu Leu Ala Arg Pro Gly Thr
Ser Val Asn Leu Ser Cys Lys Thr 35 40 45 Ser Gly Tyr Ser Phe Thr
Gly Tyr Gly Val Ser Trp Val Lys Gln Arg 50 55 60Ile Gly Gln Gly Leu
Glu Trp Ile Gly Glu Ile Tyr Pro Arg Ser Gly65 70 75 80Asn Thr Tyr
Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala 85 90 95Asp Lys
Ser Ser Asn Thr Ala Tyr Met Glu Leu Arg Ser Leu Thr Ser 100 105
110Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Trp Asn Trp Glu Arg Asp
115 120 125Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
Ser Ala 130 135 140Lys Thr Thr Pro Pro Ser Asp Tyr Pro Leu Ala Gly
Gly Gly Gly Ser145 150 155 160Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser Asp Ile Val Met Thr Gln 165 170 175Thr Pro Leu Ser Leu Pro Val
Ser Leu Gly Asp Gln Ala Ser Ile Ser 180 185 190Cys Arg Ser Ser Gln
Ser Leu Glu Ser Ser Ser Gly Tyr Pro His Leu 195 200 205Asn Trp Tyr
Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr 210 215 220Arg
Val Ser Asn Arg Phe Ser Gly Val Leu Asp Arg Phe Ser Gly Ser225 230
235 240Gly Ser Gly Thr Asp Phe Thr Leu Gln Ile Ser Arg Val Glu Ala
Glu 245 250 255Asp Leu Gly Val Tyr Phe Cys Leu Gln Ile Thr His Val
Pro Trp Thr 260 265 270Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
Ala Asp Ala Ala Pro 275 280 285Thr Val Ser Ala Ala Glu Glu Gln Lys
Leu Ile Ser Glu Glu Asp Leu 290 295 300Glu His His His His His
His305 3107821DNAMus musculus 7aaaaccatgg cggatgtgaa gcttcaggag
tctggggctg aactggtgaa gcctggggct 60tcagtgaagt tgtcctgcaa ggcttctggc
tacaccttca ccagctacta tatgtactgg 120gtgaagcaga ggcctggaca
aggccttgag tggattggag agattaatcc tagcaatggt 180ggtactaact
tcaatgagaa gttcaagagc aaggccacac tgactgtaga caaatcctcc
240agcacagcat acatgcaact cagcagcctg acatctgagg actctgcggt
ctattactgt 300acaagagact ataggtacgc ctggtttgct tactggggcc
aagggactct ggtcactgtc 360tctgcagaga gtcagtcctt cccaaatgtc
ttccccctcg ccggcggagg cggttcaggc 420ggaggtggct ctggcggtgg
cggatccgat gttttgatga cccaaactcc actctccctg 480cctgtcagtc
ttggagatca agcctccatc tcttgcagat ctagtcagag cattgtacat
540agtaatggaa acacctattt agaatggtac ctgcagaaac caggccagtc
tccaaagctc 600ctgatctaca aagtttccaa ccgattttct ggggtcccag
acaggttcag tggcagtgga 660tcagggacag atttcacact caagatcagc
agagtggagg ctgaggatct gggagtttat 720tactgctttc aaggttcaca
tgttccgtac acgttcggag gggggaccaa gctggaaata 780aaacgggctg
atgctgcacc aactgtatcc cggccgcttt t 8218480DNAMus musculus
8atgaaatacc tgctgccgac cgctgctgct ggtctgctgc tcctcgctgc ccagccggcg
60atggccatgg cggaggtcca gctgcagcag tctggagctg agctggcgag gcctgggact
120tcagtgaacc tgtcttgtaa gacttctggc tacagcttca caggctatgg
tgtaagttgg 180gtgaagcagc gaattggaca gggccttgag tggattggag
agatttatcc tagaagtggt 240aatacttact acaatgagaa gttcaagggc
aaggccacac tgactgcaga caaatcctcc 300aacacggcgt acatggagct
ccgcagcctg acatctgagg actctgcggt ctatttctgt 360gcaagatgga
actgggaaag ggatgcaatg gactactggg gtcaaggaac ctcagtcacc
420gtctcctcag ccaaaacgac acccccatct gactatccac tagccggcgg
aggcggttca 4809369DNAMus musculus 9gacattgtga tgacccaaac tccactctcc
ctgcctgtca gtcttggaga tcaagcctcc 60atctcttgca ggtctagtca gagccttgaa
agcagtagtg gatatcccca tttgaactgg 120tacctccaga aaccaggcca
gtctccacaa ctcctgatct acagggtttc caaccgattt 180tctggggtcc
tagacaggtt cagtggtagt ggatcaggga cagatttcac actgcaaatc
240agcagagtgg aggctgagga tttgggagtt tatttctgcc tccaaattac
acatgtcccg 300tggacgttcg gtggaggcac caagctggaa atcaaacggg
ctgatgctgc accaactgta 360tccgcggcc 36910936DNAArtificialscFv
Myc-His (H6/L5) 10atgaaatacc tgctgccgac cgctgctgct ggtctgctgc
tcctcgctgc ccagccggcg 60atggccatgg cggaggtcca gctgcagcag tctggagctg
agctggcgag gcctgggact 120tcagtgaacc tgtcttgtaa gacttctggc
tacagcttca caggctatgg tgtaagttgg 180gtgaagcagc gaattggaca
gggccttgag tggattggag agatttatcc tagaagtggt 240aatacttact
acaatgagaa gttcaagggc aaggccacac tgactgcaga caaatcctcc
300aacacggcgt acatggagct ccgcagcctg acatctgagg actctgcggt
ctatttctgt 360gcaagatgga actgggaaag ggatgcaatg gactactggg
gtcaaggaac ctcagtcacc 420gtctcctcag ccaaaacgac acccccatct
gactatccac tagccggcgg aggcggttca 480ggcggaggtg gctctggcgg
tggcggatcc gacattgtga tgacccaaac tccactctcc 540ctgcctgtca
gtcttggaga tcaagcctcc atctcttgca ggtctagtca gagccttgaa
600agcagtagtg gatatcccca tttgaactgg tacctccaga aaccaggcca
gtctccacaa 660ctcctgatct acagggtttc caaccgattt tctggggtcc
tagacaggtt cagtggtagt 720ggatcaggga cagatttcac actgcaaatc
agcagagtgg aggctgagga tttgggagtt 780tatttctgcc tccaaattac
acatgtcccg tggacgttcg gtggaggcac caagctggaa 840atcaaacggg
ctgatgctgc accaactgta tccgcggccg aagaacagaa actgatcagc
900gaagaagatc tcgagcacca ccaccaccac cactga 9361117PRTArtificialM13A
mutant of CDR3H 11Tyr Phe Cys Ala Arg Trp Asn Trp Glu Arg Asp Ala
Ala Asp Tyr Trp1 5 10 15Gly1217PRTArtificialD11R mutant of CDR3H
12Tyr Phe Cys Ala Arg Trp Asn Trp Glu Arg Arg Ala Met Asp Tyr Trp1
5 10 15Gly1317PRTArtificialR10A mutant of CDR3H 13Tyr Phe Cys Ala
Arg Trp Asn Trp Glu Ala Asp Ala Met Asp Tyr Trp1 5 10 15Gly
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